Mutant of terminal deoxynucleotidyl transferase, production method and application

Abstract

The invention relates to a mutant of terminal deoxynucleotidyl transferase, a production method and application. Relative to a terminal deoxynucleotidyl transferase as shown in SEQ ID NO.1, the terminal deoxynucleotidyl transferase has one or more of mutations as shown in (1) to (8): (1) mutation of glutamic acid (E) at the 47 site to threonine (T), (2) mutation of glutamic acid (E) at the 54 site to threonine (T) or serine (S), (3) mutation of lysine (K) at the 73 site to arginine (R), (4) mutation of arginine (R) at the 210 site to leucine (L), (5) mutation of lysine (K) at the 212 site to glycine (G), and (6) mutation of aspartic acid (D) at the 268 site (7) methionine (M) at the 270 site is mutated into phenylalanine (F), and phenylalanine (F) at the 273 site is mutated into leucine (L) or tyrosine (Y). Compared with a corresponding wild type, the mutant has the advantages that the doping efficiency of a modified substrate can be improved, and the enzyme activity is effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and enzyme engineering, and in particular to a mutant of terminal deoxynucleotidyl transferase, a production method and application thereof. Background Art

[0002] DNA synthesis technology mainly includes chemical method and biological method. Among them, the chemical method (especially the solid phase phosphoramidite triester synthesis method) is the most mature and widely used, while the biological method has appeared abroad, but is still in the principle verification stage.

[0003] As the length of oligonucleotide synthesis increases, the error rate of chemical methods increases, and the product yield also decreases significantly. In addition, a large amount of chemical reagents are required during the synthesis process, involving strong acids and strong oxidants. The waste liquid and waste gas generated seriously pollute the environment, resulting in high subsequent treatment costs.

[0004] In recent years, experts and scholars have turned their attention to biosynthesis methods that do not rely on chemical reagents. Among them, bioenzymatic DNA synthesis technology is usually carried out in an aqueous environment, which can effectively avoid the problems existing in chemical methods and is expected to synthesize longer DNA molecules at a lower cost.

[0005] The bioenzymatic method includes biosynthetic technologies such as the terminal deoxynucleotidyltransferase (TdT) enzymatic method, coupling method, mixed enzyme method and metal ion regulation. TdT and some DNA polymerases can directly catalyze the synthesis of DNA chains without relying on existing DNA template molecules, and combined with in vivo assembly methods such as homologous recombination, the length and accuracy of oligonucleotide synthesis can be increased by several orders of magnitude, greatly improving the ability to design and construct using synthetic biology. Compared with chemical methods for synthesizing DNA, the bioenzymatic method has great prospects and is expected to create significant value in terms of synthesis length and yield.

[0006] TdT is a template-independent enzyme that usually extends the DNA chain in a random manner and can add four natural bases to the 3' end of the DNA chain. Nucleotide monomers with reversible termination groups are chemically synthesized, and then the TdT enzyme is used to continuously add bases to the ends of the synthesized fragments. Only a single target base can be extended each time, and then the termination group is removed and the synthesis of the next target base is started. A total of two steps can complete a round of base incorporation.

[0007] However, TdT currently still has problems such as incorporation efficiency, so how to improve TdT activity from the perspective of protein modification is an urgent issue to be addressed. Summary of the invention

[0008] Based on this, one of the purposes of the embodiments of the present application includes providing a mutant of terminal deoxynucleotidyl transferase, which can improve the incorporation efficiency of the modified substrate compared with its wild type, and the enzyme activity is effectively improved.

[0009] An embodiment of the present application provides a mutant of terminal deoxynucleotidyl transferase, which has one of the mutations shown in (1) to (8) relative to the terminal deoxynucleotidyl transferase shown in SEQ ID NO.1: (1) glutamic acid (E) at position 47 mutates to threonine (T), (2) glutamic acid (E) at position 54 mutates to threonine (T) or serine (S), (3) lysine (K) at position 73 mutates to arginine (R), (4) arginine (R) at position 210 mutates to leucine (L), (5) lysine (K) at position 212 mutates to glycine (G), (6) aspartic acid (D) at position 268 mutates to serine (S), (7) methionine (M) at position 270 mutates to phenylalanine (F), and (8) phenylalanine (F) at position 273 mutates to leucine (L) or tyrosine (Y).

[0010] The embodiments of the present application also provide a nucleic acid molecule encoding the mutant of terminal deoxynucleotidyl transferase as described above.

[0011] An embodiment of the present application also provides an expression vector comprising the nucleic acid molecule as described above.

[0012] In some specific embodiments, the expression vector comprises a plasmid; optionally, the plasmid comprises a pET-28a plasmid.

[0013] An embodiment of the present application further provides a host cell, which comprises the nucleic acid molecule as described above or the expression vector as described above; optionally, the host cell comprises an Escherichia coli cell; optionally, the Escherichia coli comprises E. coli BL21 or E. coli top10.

[0014] The embodiments of the present application also provide a method for producing a mutant of terminal deoxynucleotidyl transferase, which comprises the following steps:

[0015] culturing the host cell as described above; and isolating the mutant from the resulting culture.

[0016] An embodiment of the present application provides a nucleic acid fragment synthesis kit, which includes the mutant described above, the nucleic acid molecule described above, the expression vector described above, or the host cell described above.

[0017] In some specific embodiments, the kit further comprises other synthesis reagents, and optionally, the other synthesis reagents include modified dNTP, Co2+ 、Na + , and one or more of reaction buffers; optionally, the reaction buffer comprises 45mM-55mM Tris-HCl buffer with a pH value of 7.0-7.4.

[0018] The embodiment of the present application provides a method for synthesizing a nucleic acid fragment, which uses the mutant as described above to add dNTPs to the starting chain with a 3' overhang to synthesize the nucleic acid fragment.

[0019] In some specific embodiments, during the synthesis process, the synthesis system comprises the following components in the following amounts:

[0020]

[0021] Optionally, the reaction buffer comprises 45 mM-55 mM Tris-HCl buffer with a pH value of 7.0-7.4.

[0022] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0024] Figure 1 The expression vector constructed in Example 1;

[0025] Figure 2 It is the SDS-PAGE detection diagram of TdT113, 115, 117, and 121 in Example 1;

[0026] Figure 3 It is the SDS-PAGE detection image of TdT3, TdT123, TdT128, TdT131, TdT133, TdT134, TdT135, TdT136, TdT137, TdT138, TdT139, and TdT141 in Example 1;

[0027] Figure 4 This is the SDS-PAGE detection image of the initial screening reaction products of TdT113, 115, 117, and 121 in Example 1;

[0028] Figure 5This is the SDS-PAGE detection image of the TdT128 and 133 primary screening reaction products in Example 1;

[0029] Figure 6 This is the SDS-PAGE detection image of the TdT134 primary screening reaction product in Example 1;

[0030] Figure 7 This is an SDS-PAGE detection diagram of the initial screening reaction products of TdT123, TdT135, TdT136, TdT137, TdT138, TdT139, and TdT141 in Example 1;

[0031] Figure 8 This is the SDS-PAGE detection image of the TdT131 primary screening reaction product in Example 1;

[0032] Fig. 9 This is the SDS-PAGE detection image of the TdT3 primary screening reaction product in Example 1;

[0033] Fig.10 This is the SDS-PAGE detection image of the TdT131 multiple screening reaction product in Example 1 (the starting chain is C / G / 2C terminal);

[0034] Fig.11 This is the SDS-PAGE detection image of the TdT113, 115, and 117 multiple screening reaction products in Example 1 (the starting chain is the C-terminus);

[0035] Fig.12 This is the SDS-PAGE detection image of the TdT113 rescreening reaction product in Example 1 (the starting chain is G / 2C terminal);

[0036] Fig.13 This is the SDS-PAGE detection image of the TdT133 multiple screening reaction product in Example 1 (the starting chain is the C-terminus);

[0037] Fig.14 This is the SDS-PAGE detection image of the TdT133 multiple screening reaction product in Example 1 (the starting chain is G / 2C terminus). DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0040] the term

[0041] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0042] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0043] In the present invention, "plurality", "multiple", "multiple times", "multiple" and the like, unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0044] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0045] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0046] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0047] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0048] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0049] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0050] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0051] In the present invention, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0052] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0053] In the present invention, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0054] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0055] An embodiment of the present application provides a mutant of terminal deoxynucleotidyl transferase, which has one of the mutations shown in (1) to (8) relative to the terminal deoxynucleotidyl transferase shown in SEQ ID NO.1: (1) glutamic acid (E) at position 47 mutates to threonine (T), (2) glutamic acid (E) at position 54 mutates to threonine (T) or serine (S), (3) lysine (K) at position 73 mutates to arginine (R), (4) arginine (R) at position 210 mutates to leucine (L), (5) lysine (K) at position 212 mutates to glycine (G), (6) aspartic acid (D) at position 268 mutates to serine (S), (7) methionine (M) at position 270 mutates to phenylalanine (F), and (8) phenylalanine (F) at position 273 mutates to leucine (L) or tyrosine (Y).

[0056] The embodiments of the present application also provide a nucleic acid molecule encoding the mutant of terminal deoxynucleotidyl transferase as described above.

[0057] An embodiment of the present application also provides an expression vector comprising the nucleic acid molecule as described above.

[0058] In some specific embodiments, the expression vector comprises a plasmid; optionally, the plasmid comprises a pET-28a plasmid.

[0059] An embodiment of the present application further provides a host cell, which comprises the nucleic acid molecule as described above or the expression vector as described above; optionally, the host cell comprises an Escherichia coli cell; optionally, the Escherichia coli comprises E. coli BL21 or E. coli top10.

[0060] The embodiments of the present application also provide a method for producing a mutant of terminal deoxynucleotidyl transferase, which comprises the following steps:

[0061] culturing the host cell as described above; and isolating the mutant from the resulting culture.

[0062] An embodiment of the present application provides a nucleic acid fragment synthesis kit, which includes the mutant described above, the nucleic acid molecule described above, the expression vector described above, or the host cell described above.

[0063] In some specific embodiments, the kit further comprises other synthesis reagents, and optionally, the other synthesis reagents include modified dNTP, Co 2+ 、Na +, and one or more of reaction buffers; optionally, the reaction buffer includes 45mM-55mM (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55mM) Tris-HCl buffer, with a pH of 7.0-7.4 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4).

[0064] The embodiment of the present application provides a method for synthesizing a nucleic acid fragment, which uses the mutant as described above to add dNTPs to the starting chain with a 3' overhang to synthesize the nucleic acid fragment.

[0065] In some specific embodiments, during the synthesis process, the synthesis system comprises the following components in the following amounts:

[0066]

[0067] And a reaction buffer; optionally, the reaction buffer comprises 45mM-55mM (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55mM) Tris-HCl buffer, with a pH value of 7.0-7.4 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4).

[0068] It will be appreciated that the host cell is cultured under culture conditions which allow the expression of the nucleic acid molecule encoding the mutant.

[0069] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0070] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0071] Example 1

[0072] 1. Methods and Results

[0073] 1.1 Gene design and synthesis

[0074] In order to improve the incorporation efficiency of TdT into the modified substrate, the structure of wild-type TdT was analyzed, and the amino acid sites glutamic acid (E) at position 47, glutamic acid (E) at position 54, lysine (K) at position 73, phenylalanine (F) at position 130, aspartic acid (D) at position 268, methionine (M) at position 270, phenylalanine (F) at position 273, phenylalanine (F) at position 277, phenylalanine (F) at position 334, and arginine (R) at position 386 were optimized.

[0075] In order to improve the catalytic effect of TdT on the modified substrate, the key amino acids adjacent to the modified substrate in the TdT structure were mutated as follows while keeping the secondary structure unchanged: glutamic acid (E) at position 47 was mutated to threonine (T), glutamic acid (E) at position 54 was mutated to threonine (T) / serine (S), lysine (K) at position 73 was mutated to arginine (R), phenylalanine (F) at position 130 was mutated to tyrosine (Y), arginine (R) at position 210 was mutated to leucine (L), lysine (K) at position 212 was mutated to glycine (G), and phenylalanine (F) at position 130 was mutated to tyrosine (Y). Aspartic acid (D) at position 8 was mutated to serine (S), methionine (M) at position 270 was mutated to phenylalanine (F), phenylalanine (F) at position 273 was mutated to leucine (L) / tyrosine (Y), phenylalanine (F) at position 277 was mutated to leucine (L) / tyrosine (Y) / isoleucine (I), phenylalanine (F) at position 334 was mutated to tyrosine (Y), and arginine (R) at position 386 was mutated to glutamine (Q) / proline (P), resulting in the following 15 mutants of wild-type TdT3, which are denoted as: TdT113, TdT2114, TdT3115, TdT3116, TdT3117, TdT3118, TdT3119, TdT3120, TdT3121. 115, TdT 117, TdT 121, TdT 123, TdT 128, TdT 131, TdT 133, TdT 134, TdT 135, TdT 136, TdT 137, TdT 138, TdT139, TdT 141.

[0076] Table 1

[0077]

[0078]

[0079] After the TdT mutant was designed, the gene was synthesized (by Nanjing Qingke Biotechnology Co., Ltd.), codon optimized and connected to the pET-28a plasmid to obtain the expression vector of the TdT mutant. At the same time, the expression vector of the TdT wild type was constructed. Figure 1 .

[0080] 1.2 Inducible expression

[0081] Extract the expression plasmids of TdT wild type and its mutant from E.coli top10, and take 5-10μL and slowly add them to E.coli BL21 (DE3) competent cells, mix well, ice bath for 30min, heat shock at 42℃ for 45-90s, and ice bath for 1-2min. Add 900μL LB, incubate at 37℃ and 220rpm for 1h, centrifuge at 5000rpm for 3min, keep a little supernatant, blow and mix, spread on LB plate medium containing kanamycin resistance, and invert and culture at 37℃ overnight. Pick the above single clones and inoculate them into 5mL LB medium containing kanamycin resistance, culture at 220rpm at 37℃ overnight, inoculate the bacterial liquid into 100mL LB medium containing kanamycin resistance at 1% inoculation, culture at 37℃ and 220rpm until OD 0.6-0.8, add IPTG with a final concentration of 0.5mM, and induce at 16℃ and 120rpm overnight.

[0082] 1.3 Protein purification

[0083] 1.3.1 Cell disruption

[0084] After the induction expression is completed, take 40mL of bacterial solution and centrifuge at 3500rpm for 15min, discard the supernatant, resuspend with 1.5mL of equilibrium buffer, add protease inhibitors at a final concentration of 1mM, and use a high-throughput non-contact ultrasonic disruptor to lyse the cells. The disruption temperature is 4°C, the power is 100%, the interval time is 3s, and the total time is 60min. After disruption, centrifuge at 12000rpm for 15min at 4°C and collect the supernatant.

[0085] 1.3.2 Magnetic bead preparation and equilibration

[0086] Take 150 μL of magnetic bead suspension and place it on a magnetic separator. When the solution becomes clear, use a pipette to discard the clear solution. Add 200 μL of equilibration buffer, pipette repeatedly for 5-10 times, place it on a magnetic separator, use a pipette to discard the clear solution, and repeat the washing for 2 times.

[0087] 1.3.3 Magnetic beads binding target protein

[0088] Add the supernatant after centrifugation to the treated magnetic beads and mix them upside down. Invert at 40 rpm for 1 hour at 4°C, then centrifuge to remove the supernatant, remove the centrifuge tube from the magnetic separator for washing. Add 400 μL of washing solution to the centrifuge tube, use the tip of the gun to blow repeatedly 5-10 times, remove the supernatant, and repeat once. Add 50-100 μL of elution solution to the centrifuge tube, invert at 40 rpm for 10 minutes at 4°C, and then centrifuge to obtain the target protein.

[0089] 1.4SDS-PAGE

[0090] Use 4%-20% SDS-PAGE precast gel (provided by Hubei Qingke Biotechnology Co., Ltd.) to put it into the electrophoresis tank for gel running, take different TdT mutant proteins, add loading buffer and mix well, load 20μL of sample, 5μL of marker, run the gel at 160V for 30min, heat and stain with dye solution for 15min, take pictures with gel imager after cooling, use the marker band size as a reference to analyze whether the size of the target protein band is correct.

[0091] Figure 2 This is the SDS-PAGE detection diagram of TdT113, TdT115, TdT117, and TdT121. Figure 3 These are SDS-PAGE detection pictures of TdT3, TdT123, TdT128, TdT131, TdT133, TdT134, TdT135, TdT136, TdT137, TdT138, TdT139 and TdT141. The SDS-PAGE results show that the target protein can be obtained after magnetic bead purification and the proportion of the target protein is high. This method can be used to purify TdT wild type and its mutants.

[0092] 1.5 Protein concentration determination

[0093] After washing the SAM4000 probe twice with ultrapure water, select the interface for protein concentration determination and 280 Use elution buffer to adjust the wavelength to zero, then take a small amount of eluted protein solution for determination, and read the concentration of the purified target protein and the 260 / 280 value. If the 260 / 280 value is too high, it indicates that the protein sample is seriously contaminated with nucleic acid, which may affect the functional verification experiment and requires re-purification of the protein.

[0094] 1.6 TdT activity detection

[0095] 1.6.1 Preliminary screening of TdT-catalyzed base incorporation reactions

[0096] The concentration of wild-type TdT and its mutants was uniformly diluted to 0.06 mg / mL, 3'-ONH2-dATP was used as the substrate, and the reaction system was prepared on ice. The specific formula is shown in the following table.

[0097] Table 2

[0098] Components Concentration (total volume 50 μL) Starting chain 1μM <![CDATA[3’-ONH2-dATP]]> 0.25mM <![CDATA[CoCl2]]> 0.25mM NaCl 100mM Tris-HCl pH 7.2 Take 2.5 μL of 50mM, 1M Tris-HCl stock solution TdT 0.06mg / mL <![CDATA[H2O]]> Make up to 50 μL

[0099] Reaction conditions: 30°C for 30 seconds, heating at 95°C for 10 minutes.

[0100] In the above TdT wild type and its mutant screening reactions, TdT3, TdT113, TdT115, TdT117, TdT121, TdT128, TdT131, TdT133, and TdT134 catalyzed the incorporation of the modified substrate into the starting chain, respectively. The results are as follows:

[0101] Figure 4 This is the SDS-PAGE detection diagram of the initial screening reaction products of TdT113, TdT115, TdT117, and TdT121;

[0102] Figure 5 This is the SDS-PAGE detection image of the initial screening reaction products of TdT128 and TdT133;

[0103] Figure 6 This is the SDS-PAGE detection image of the TdT134 primary screening reaction product;

[0104] Figure 7 This is the SDS-PAGE detection diagram of the initial screening reaction products of TdT123, TdT135, TdT136, TdT137, TdT138, TdT139, and TdT141;

[0105] Figure 8 This is the SDS-PAGE detection image of the TdT131 primary screening reaction product;

[0106] Fig. 9 This is the SDS-PAGE detection diagram of the TdT3 initial screening reaction product. The SDS-PAGE results show that the incorporation rate of TdT3 is about 50%; TdT123, TdT135, TdT136, TdT137, TdT138, TdT139, and TdT141 can hardly be incorporated; the incorporation efficiency of TdT121 is 80%; TdT113, TdT115, TdT117, TdT128, TdT131, and TdT133 can be fully incorporated, and the incorporation efficiency is more than 99%, so TdT113, TdT115, TdT117, TdT131, and TdT133 were rescreened.

[0107] Table 3

[0108]

[0109] 1.6.2 TdT-catalyzed base incorporation reaction rescreening

[0110] The concentration of wild-type TdT and its mutants was uniformly diluted to 0.5 mg / mL, 3'-ONH2-dA(T / C / G)TP was used as substrate, and starting chains with different base ends were selected. The reaction system was prepared on ice. The specific formula is shown in the following table.

[0111] Table 4

[0112] Components Concentration (total volume 50 μL) Starting chain 1μM <![CDATA[3’-ONH2-dA(T / C / G)TP]]> 0.25mM <![CDATA[CoCl2]]> 0.25mM NaCl 100mM Tris-HCl pH7.2 50mM TdT 0.5mg / mL <![CDATA[H2O]]> Make up to 50 μl

[0113] Reaction conditions: 30℃ for 30s, 95℃ for 10min. The results are shown in the figure below:

[0114] Fig.10 This is the SDS-PAGE detection image of TdT131 rescreening reaction product (the starting chain is C / G / 2C terminal);

[0115] Fig.11 This is the SDS-PAGE detection diagram of the rescreening reaction products of TdT113, TdT115, and TdT117 (the starting chain is the C-terminus);

[0116] Fig.12 This is the SDS-PAGE detection image of TdT113 rescreening reaction product (the starting chain is G / 2C terminal);

[0117] Fig.13 This is the SDS-PAGE detection image of TdT133 rescreening reaction product (the starting chain is the C-terminus);

[0118] Fig.14 This is the SDS-PAGE detection image of TdT133 rescreening reaction product (the starting chain is G / 2C terminal);

[0119] In the figure: CA represents the incorporation efficiency of A when the starting chain ends at C; CT represents the incorporation efficiency of T when the starting chain ends at C; and so on. In the figures of this embodiment:

[0120] Control 21nt (SEQ ID NO. 18): TTTTTTTTTTTTTTTTTTTTT.

[0121] Control 22nt (SEQ ID NO. 19): TTTTTTTTTTTTTTTTTTCC.

[0122] Control 23nt (SEQ ID NO. 20): TTTTTTTTTTTTTTTTTTTTTTT.

[0123] When the C-terminal initiator chain incorporates modified substrates, TdT131 is almost unable to incorporate 3'-ONH2-dATP, and has only about 50% incorporation efficiency for the three modified substrates 3'-ONH2-dT(C / G)TP; TdT115 and TdT117 are unable to incorporate; while TdT113 and TdT133 are able to incorporate the four modified substrates 3'-ONH2-dA(T / C / G)TP.

[0124] When the G-terminated initiator chain was incorporated into the modified substrate, TdT131 could almost completely react to the four modified substrates of 3'-ONH2-dA(T / C / G)TP; TdT113 and TdT133 could incorporate the four modified substrates of 3'-ONH2-dA(T / C / G)TP.

[0125] When the starting chain with 2C as the end was incorporated into modified substrates, TdT131 was almost unable to incorporate any modified substrates; TdT113 was able to incorporate four modified substrates of 3'-ONH2-dA(T / C / G)TP; TdT133 was able to incorporate 50% of 3'-ONH2-dATP and about 80% of the three modified substrates of 3'-ONH2-dT(C / G)TP.

[0126] In the repeated screening reactions of TdT113, TdT115, TdT117, TdT131, and TdT133, TdT113 showed very high modified substrate incorporation activity.

[0127] Table 5

[0128]

[0129]

[0130] The starting chain is the C-terminal: TTTTTTTTTTTTTTTTTTTTTC (SEQ ID NO. 21).

[0131] Corresponding product: TTTTTTTTTTTTTTTTTTTTTTCT (SEQ ID NO. 23).

[0132] The starting chain is G-terminal: TTTTTTTTTTTTTTTTTTTTTG (SEQ ID NO. 22).

[0133] Corresponding product: TTTTTTTTTTTTTTTTTTTTTTGT (SEQ ID NO. 24).

[0134] The starting chain is 2C-terminal: TTTTTTTTTTTTTTTTTTTTCC (SEQ ID NO. 19).

[0135] Corresponding product: TTTTTTTTTTTTTTTTTTTTTCCT (SEQ ID NO. 25).

[0136] 1.6.3SDS-PAGE

[0137] Prepare 20% SDS-PAGE separation gel and put it into the electrophoresis tank for running after the gel solidifies. The specific formula of the separation gel is shown in the following table.

[0138] Table 6

[0139] Components Volume (mL) <![CDATA[H2O]]> 0.84 30% Acr-Bis (29:1) 6.66 SDS-PAGE Separating Gel Buffer(4×) 2.5 10% APS 0.1 TEMED 0.004 Total volume 10

[0140] The reaction products of different TdT mutants were added to 2×Loading buffer and mixed, and 5 μL was loaded. The oligonucleotides in the control group were added to 2×Loading buffer and mixed, and 5 μL was loaded. The gel was run at 220V for 90 min, and photos were taken using a gel imager. Oligonucleotide chains of different lengths, 20nt, 21nt, and 22nt, were used as controls to analyze the oligonucleotide extension length and the incorporation efficiency of the modified substrate, and TdT mutants with better incorporation effects were screened.

[0141] 2. Analysis and discussion

[0142] In the initial screening reaction of TdT and its mutants, TdT3, TdT113, TdT115, TdT117, TdT121, TdT128, TdT131, TdT133 and TdT134 catalyzed the incorporation of the starting chain into the modified substrate respectively. SDS-PAGE results showed that TdT3 could not catalyze the complete incorporation of the modified substrate, with an incorporation rate of about 50%. TdT113, TdT115, TdT117, TdT131 and TdT133 could be completely incorporated, with an incorporation efficiency of more than 99%. Therefore, TdT113, TdT115, TdT117, TdT131 and TdT133 were rescreened.

[0143] Among them, TdT113 can react completely in the primary screening, and the initial chains with C / G / 2C as the ends can be incorporated with the modified substrates in the rescreening; TdT115 can also react completely in the primary screening, but the initial chain with C as the end does not react at all in the rescreening; TdT117 can also react completely in the primary screening, but the modified substrate cannot be completely incorporated in the initial chain with C as the end in the rescreening, and the incorporation rate is about 50%; TdT121 cannot be completely incorporated in the primary screening, and the incorporation rate is about 80%; TdT128 reacts almost completely in the primary screening, and the unreacted band is almost invisible, but the initial chain with C as the end does not react at all in the rescreening; TdT131 can react completely in the primary screening, but the initial chain with C as the end in the rescreening , 3'-ONH2-dATP did not react at all, 3'-ONH2-dT(C / G)TP did not react completely, and the starting chain with 2C as the end did not react at all; TdT133 could react completely in the initial screening and the starting chain with C or G as the end could be incorporated with the modified substrate in the rescreening, but when the initial chain with 2C as the end was incorporated with the modified substrate, TdT133 could incorporate 50% of 3'-ONH2-dATP and about 80% of the three modified substrates of 3'-ONH2-dT(C / G)TP; TdT134 reacted almost completely in the initial screening, with slightly more unreacted bands than TdT128, but the starting chain with C as the end did not react at all in the rescreening.

[0144] In summary, TdT113 exhibits very high modified substrate incorporation activity, especially the reaction of modified substrates that are more difficult to incorporate can be basically catalyzed to completion, with relatively small preference.

[0145] The amino acid sequence of TdT 3 (wild type) is shown in SEQ ID NO.1:

[0146] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRD IIEEIIIEGEESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAAEADAVSLIVKNTVC TFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0147] The amino acid sequence of TdT 113 is shown in SEQ ID NO. 2 (the 47th glutamic acid (E) is mutated to threonine (T)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAF VMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDI DFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0148] The amino acid sequence of TdT 115 is shown in SEQ ID NO.3 (glutamic acid (E) at position 54 is mutated to threonine (T)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENY FKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFL ITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0149] The amino acid sequence of TdT 117 is shown in SEQ ID NO.4 (glutamic acid (E) at position 54 is mutated to serine (S)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENY FKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA。

[0150] The amino acid sequence of TdT 121 is shown as SEQ ID NO.5 below (lysine (K) at position 73 is mutated to arginine (R)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLL SLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA。

[0151] The amino acid sequence of TdT 123 is shown in SEQ ID NO.6 (phenylalanine (F) at position 130 is mutated to tyrosine (Y)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSV GVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVK EQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHGLDYVEPWERNA.

[0152] The amino acid sequence of TdT 128 is shown in SEQ ID NO.7 (the aspartic acid (D) at position 268 is mutated to serine (S)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKV AMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHGLDYVEPWERNA.

[0153] The amino acid sequence of TdT 131 is shown in SEQ ID NO.8 (the methionine (M) at position 270 is mutated to phenylalanine (F)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDA DHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHGLDYVEPWERNA.

[0154] The amino acid sequence of TdT 133 is shown in SEQ ID NO.9 (phenylalanine (F) at position 273 is mutated to leucine (L)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDH QKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0155] The amino acid sequence of TdT 134 is shown in SEQ ID NO.10 (phenylalanine (F) at position 273 is mutated to tyrosine (Y)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDH QKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0156] The amino acid sequence of TdT 135 is shown in SEQ ID NO.11 (phenylalanine (F) at position 277 is mutated to leucine (L)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKC AILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHGLDYVEPWERNA.

[0157] The amino acid sequence of TdT 136 is shown in SEQ ID NO.12 below (phenylalanine (F) at position 277 is mutated to tyrosine (Y)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKC AILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHGLDYVEPWERNA.

[0158] The amino acid sequence of TdT 137 is shown in SEQ ID NO. 13 (phenylalanine (F) at position 277 is mutated to isoleucine (I)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKC AILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHGLDYVEPWERNA.

[0159] The amino acid sequence of TdT 138 is shown in SEQ ID NO.14 below (phenylalanine (F) at position 334 is mutated to tyrosine (Y)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQ GRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0160] The amino acid sequence of TdT 139 is shown in SEQ ID NO. 15 (arginine (R) at position 386 is mutated to glutamine (Q)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCV SKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWE NA.

[0161] The amino acid sequence of TdT 141 is shown in SEQ ID NO. 16 (the arginine (R) at position 386 is mutated to proline (P)): MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCV SKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWE NA.

[0162] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0163] The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solutions of the present invention in detail, but cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. A terminal deoxynucleotidyl transferase mutant having one of the mutations (1) to (8) relative to the terminal deoxynucleotidyl transferase shown in SEQ ID NO.1: (1) The glutamic acid (E) at position 47 was mutated to threonine (T), (2) The glutamic acid (E) at position 54 is mutated to threonine (T) or serine (S), (3) Lysine (K) at position 73 mutated to arginine (R), (4) Arginine (R) at position 210 mutated to leucine (L), (5) Lysine (K) at position 212 was mutated to glycine (G), (6) Aspartic acid (D) at position 268 mutated to serine (S), (7) mutation of methionine (M) at position 270 to phenylalanine (F), and (8) The phenylalanine (F) at position 273 mutates to leucine (L) or tyrosine (Y).

2. A nucleic acid molecule encoding a mutant of terminal deoxynucleotidyl transferase as described in claim 1. An expression vector comprising the nucleic acid molecule according to claim 2 .

4. The expression vector according to claim 3, comprising a plasmid; optionally, the plasmid comprises a pET-28a plasmid.

5. A host cell comprising the nucleic acid molecule of claim 2 and the expression vector of claim 3 or 4; optionally, the host cell comprises an Escherichia coli cell; optionally, the Escherichia coli comprises E. coli BL21 or E. coli top10.

6. A method for producing a mutant of terminal deoxynucleotidyl transferase, comprising the following steps: Cultivating the host cell according to claim 5; and, Mutants were isolated from the resulting cultures.

7. A nucleic acid fragment synthesis kit, comprising the mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3 or 4, or the host cell according to claim 5.

8. The kit according to claim 7, further comprising other synthetic reagents, optionally, the other synthetic reagents comprising modified dNTP, Co 2+ 、Na + , and one or more of reaction buffers; optionally, the reaction buffer comprises 45mM-55mM Tris-HCl buffer with a pH value of 7.0-7.

4.

9. A method for synthesizing a nucleic acid fragment, comprising using the mutant according to claim 1 to add dNTP to a starting chain having a 3' overhang to synthesize the nucleic acid fragment.

10. The method for synthesizing a nucleic acid fragment according to claim 9, wherein during the synthesis process, the synthesis system comprises the following amounts of components: and reaction buffer; Optionally, the reaction buffer comprises 45 mM-55 mM Tris-HCl buffer with a pH value of 7.0-7.4.

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